Cellular and Molecular Response to Uranium
and Depleted Uranium Exposure
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Summary
The papers in this chapter show that uranium (in various forms) does cause chromosomal damage and genetic aberrations in cells, such as increases in sister chromatid exchanges and micronuclei formation, which are indicative of DNA strand breaks. These changes are often precursors to carcinogenesis. Increased strand breaks in germ cells (sperm and ova) can lead to greater risk of birth defects in offspring, a mutagenic effect, and direct effects on the developing fetus, a teratogenic effect. These combined effects are referred to as cytogenetic toxicity. Another common effect observed with radiation exposure is genomic instability. This occurs when surviving irradiated cells are found to also exhibit functional abnormalities. Depleted uranium exposure has been shown to cause a significant effects lasting through as many as 30 generations of progeny cells. Finally, the bystander effect is observed when cells surrounding those that have been directly irradiated are also found to exhibit abnormalities.
Details
Cytotoxicity of uranium in rat lung tissue was reported as early as 1987 by Tasat (1). In 1993 Lin showed uranium compounds to possess cytogenetic toxicity and cause decreased cell viability in hamster ovary cells, explaining the teratogenic effects on developing fetal mice (2) observed and reported by Domingo in 1989.
Alexandria Miller’s continuing work at the Armed Forces Radiobiology Institute in Bethesda has shown that uranium exposure transforms human osteoblast cells in vitro to a tumorigenic phenotype (3, 9), implying that internalized DU exposure would be biologically active and could lead to cancer, similar to exposure to other heavy metals such as nickel and tungsten (5), but more potent (7). She demonstrated that phenyl acetate, a chemotherapeutic agent, showed some tendancy to suppress these transformation effects of DU (6). In comparison with nickel and tungsten, neither of which are radioactive, she demonstrated that DU exposure resulted in significantly increased dicentric frequency, a radiation induced genotoxic effect, that was radiation-dose dependant (8). She also demonstrated that DU exposure produces a significant genomic instability effect that lasted three times longer (through 30 successive generations) than that for gamma radiation or for nickel exposure and that the affected progeny cells exhibited considerably more chromosomal damage than did progeny cells whose precurser cells were exposed to gamma radiation, while the nickel-exposed ancestor cells produced progeny with no elevated level of micronuclei formation (11). Her most recent work tested the ability of DU and metals in a typical tungsten alloy to induce stress genes in 13 different recombinant cell lines generated from human liver carcinoma cells, with the result that both DU and metal components of tungsten alloys activate gene expression through pathways that may be involved in the toxicity and tumorigenicity of these metals (14).
Prabhavathi has shown that smokers at a nuclear fuel manufacturing facility had considerably more signs of chromosomal damage than smokers and nonsmokers who were not exposed to radiation (4). Inhaled or embedded DU is known to become associated with macrophages. Kalinich reported finding that cell death (apoptosis) occurred in a line of mouse macrophages that were exposed to DU (10). He also noted other morphological changes and DNA fragmentation in the exposed cells. Yazze ran in-vitro studies on the effects of of uranyl acetate/ascorbic acid mixtures on cells and observed plasmid relaxation responses in pBluescript DNA leading to DNA strand cleavage, suggesting that uranium, like chromium, may be directly genotoxic (13). The effect observed increased as uranium concentration increased, and was inhibited in the presence of catalase. Free-radical scavangers showed no effect.
Schroder analyzed blood lymphocytes samples from 16 veterans of war theaters where DU was used and discovered in each sample a statistically significant increased frequency of dicentric chromosomes and centric ring chromosomes, compared to samples from unexposed controls (12). These chromosomal defects indicate a previous exposure of the veterans to ionising radiation . Prat (15) has identified genetic biomarkers for uranium exposure and specific cellular response to uranium exposure.
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1. Cytotoxic effect of uranium dioxide on rat alveolar macrophages, by DR Tasat, et al., Environmental Research Vol. 44, 1987 (pp. 71-81).
[Tasat1987xxERv44nxp71].
2. Cytogenetic toxicity of uranyl nitrate in Chinese hamster
ovary cells, by R.H. Lin, et al., Mutation Research Vol. 319,
1993 (pp. 197-203).
Uranyl nitrate decreased the viability of CHO cells in
a dose-related fashion, with IC50 (conc for 50% inhibition) was 0.049 mM. At
0.01 to 0.3 mM uranyl nitrate decreased cell cycle kinetics, increased frequency
of micronuclei and sister chromatid exchange and augmented chromosomal
aberrations. Results indicates that uranyl nitrate causes genotoxicity
and cytotoxicity in CHO cells and provides the biochemical basis for
teratogenic effect of U on developing fetal mice (Domingo et al, Toxicology,
55, 143-152, 1989).
[Lin1993xxMRv319nxp197]
3. Transformation of human osteoblast cells to the
tumorigenic phenotype by depleted uranium-uranyl chloride, by A. Miller,
et al., Applied Cellular Radiobiology Department, Armed Forces Radiobiology
Research Institute,
Depleted uranium (DU) is a dense heavy metal used
primarily in military applications. Although the health effects of occupational
uranium exposure are well known, limited data exist regarding the long-term
health effects of internalized DU in humans. We established an in vitro
cellular model to study DU exposure. Microdosimetric assessment, determined
using a
[Miller199808EHPv106n8p465]. (PMID: 9681973 [PubMed -
indexed for MEDLINE]).
4. Analysis of chromosomal aberration frequencies in the peripheral blood lymphocytes of smokers exposed to uranyl compounds, by P.A. Prabhavathi, et al., Mutation Research Vol. 466, 2000 (pp. 37-41).
Studied 115 workers at a nuclear fuel manufacturing
facility (in
[Prabhavathi2000xxMRv466nxp37].
5. Neoplastic
transformation of human osteoblast cells to the tumorigenic phenotype by heavy
metal-tungsten alloy particles: induction of genotoxic effects, by A.
Miller, et al., Applied Cellular Radiobiology Department, Armed Forces
Radiobiology Research Institute,
Heavy metal-tungsten alloys (HMTAs) are dense heavy
metal composite materials used primarily in military applications. HMTAs are
composed of a mixture of tungsten (91-93%), nickel (3-5%) and either cobalt
(2-4%) or iron (2-4%) particles. Like the heavy metal depleted uranium (DU),
the use of HMTAs in military munitions could result in their internalization in
humans. Limited data exist, however, regarding the long-term health effects of
internalized HMTAs in humans. We used an immortalized, non-tumorigenic, human
osteoblast-like cell line (HOS) to study the tumorigenic transforming potential
of reconstituted mixtures of tungsten, nickel and cobalt (rWNiCo) and tungsten,
nickel and iron (rWNiFe). We report the ability of rWNiCo and rWNiFe to
transform immortalized HOS cells to the tumorigenic phenotype. These HMTA
transformants are characterized by anchorage-independent growth, tumor
formation in nude mice and high level expression of the K-ras oncogene.
Cellular exposure to rWNiCo and rWNiFe resulted in 8.90 +/- 0.93- and 9.50 +/-
0.91-fold increases in transformation frequency, respectively, compared with
the frequency in untreated cells. In comparison, an equivalent dose of
crystalline
[Miller200101Cv22n1p115]. (PMID: 11159749 [PubMed -
indexed for MEDLINE]).
6. Suppression of depleted uranium-induced neoplastic transformation of human cells by the phenyl fatty acid, phenyl acetate: chemoprevention by targeting the p21RAS protein pathway, by A.C. Miller, et al., Radiation Research Vol. 155, 2001 (pp. 163-170).
This study was done as a follow up to a previous study
[Miller et al., Envir. Health Persp.106, 465-471, 1998, above] that shows
transformation of human cells to tumorigenic phenotype following exposure to
U. This paper shows that phenyl acetate (a potential chemotherapeutic
agent) could prevent transformation to the tumorigenic phenotype and decreased
membrane associated p21RAS protein, an inducer of cell transformation.
Phenyl acetate can interfere with p21 processing by blocking the mevalonate
pathway and farnesol synthesis. Farnesylation of p21 results in its
membrane association. The authors also speculate that uranyl ions may
catalyze free radical that could be involved in transformation. They do
not mention possible effects that phenyl acetate might have on the bystander
effect (see following section).
[Miller2001xxRRv155nxp163].
7. Potential
late health effects of depleted uranium and tungsten used in armor-piercing
munitions: comparison of neoplastic transformation and genotoxicity with the
known carcinogen nickel,
by A. Miller, et al., Applied Cellular Radiobiology Department
Armed Forces Radiobiology Research Institute,
Limited data exist to permit an accurate assessment of
risks for carcinogenesis and mutagenesis from embedded fragments or inhaled
particulates of depleted uranium (DU). Ongoing studies have been designed to
provide information about the carcinogenic potential of DU using in-vitro and
in-vivo assessments of morphological transformation as well as cytogenetic,
mutagenic, and oncogenic effects. For comparison, we also examined tungsten
alloys used in military projectiles and the known carcinogen nickel.
Quantitative and qualitative in-vitro transformation studies were done to
assess the carcinogenic potential of radiation and chemical hazards. Using a
human cell osteosarcoma cell model, we demonstrated that soluble and insoluble
DU compounds can transform cells to the tumorigenic phenotype, as characterized
by morphological, biochemical and oncogenic changes consistent with tumor cell
behavior. Tungsten alloys and nickel were also shown to be neoplastic
transforming agents, although at a frequency less than that of DU. Sister
chromatid exchange, micronuclei, and alkaline filter elution assays showed DU
and tungsten alloys were genotoxic. Exposure to a non-toxic, nontransforming
dose of DU induced a small but statistically significant increase in the number
of dicentrics formed in cells. These results suggest that long term exposure to
DU or tungsten alloys could be critical to the development of neoplastic
disease in humans and that additional studies are needed.
[Miller200202MMv167n2p120]. ( PMID: 11873492 [PubMed -
indexed for MEDLINE]).
8. Observation of radiation-specific damage in
human cells exposed to depleted uranium: dicentric frequency and neoplastic
transformation as endpoints, by
A Miller, et al., Applied Cellular
Radiobiology Department Armed Forces Radiobiology Research Institute,
Depleted uranium (DU) is a dense heavy metal used
primarily in military applications. Published data from our laboratory have demonstrated
that DU exposure in-vitro to immortalsed human osteoblast cells (HOS) is both
neoplastically transforming and genotoxic. DU possesses both a radiological
(alpha particle) and chemical (metal) component. Since DU has a low specific
activity in comparison to natural uranium, it is not considered to be a
significant radiolgical hazard. The potential contribution of radiation to
DU-induced biological effects is unknown and the involvement of radiation in
DU-induced biological effects could have significant implications for current
risk estimates for internalised DU exposure. Two approaches were used to
address this question. The frequency of dicentrics was measured in HOS cells
following DU exposure in vitro. Data demonstrated that DU exposure (50 micorM,
24h) induced a significant elevation in dicentric frequency in vitro in
contrast to incubation with heavy metals, nickel and tungsten, which did not
increase dicentric frequency above background levels. Using the same
concentration (50 microM) of three uranyl nitrate compounds that have different
uranium isotopic concentrations and therefore different specific activities,
the effect on neoplastic transformation in-vitro was examined. HOS cells were
exposed to one of three-uranyl nitrate compounds (238U-uranyl nitrate, specific
activity 0.33 microCi.g-1; DU uranyl nitrate, specific activity 0.44
microCi.g-1; and 235U-uranyl nitrate, specific activity 2.2 microCi.g-1)
delivered at a concentration of 50 microM for 24 h. Results showed, at equal
uranium concentration, there was a specific activity dependent increase in
neoplastic transformation frequency. Taken together, these data suggest that
radiation can play a role in DU-induced biological effects in vitro.”
[Miller200201RPDv99n1p275]. (PMID: 12194305 [PubMed -
indexed for MEDLINE]).
9. Depleted Uranium-catalyzed
oxidative DNA damage: absence of significant alpha particle decay, by A.
Miller, et al., Applied Cellular Radiobiology Department Armed Forces
Radiobiology Research Institute,
Depleted
uranium (DU) is a dense heavy metal used primarily in military applications.
Published data from our laboratory have demonstrated that DU exposure in vitro
to immortalized human osteoblast cells (HOS) is both neoplastically
transforming and genotoxic. DU possesses both a radiological (alpha particle)
and a chemical (metal) component. Since DU has a low-specific activity in
comparison to natural uranium, it is not considered to be a significant
radiological hazard. In the current study we demonstrate that DU can generate
oxidative DNA damage and can also catalyze reactions that induce hydroxyl
radicals in the absence of significant alpha particle decay. Experiments were
conducted under conditions in which chemical generation of hydroxyl radicals
was calculated to exceed the radiolytic generation by 10(6)-fold. The data
showed that markers of oxidative DNA base damage, thymine glycol and
8-deoxyguanosine could be induced from DU-catalyzed reactions of hydrogen
peroxide and ascorbate similarly to those occurring in the presence of iron
catalysts. DU was 6-fold more efficient than iron at catalyzing the oxidation
of ascorbate at pH 7. These data not only demonstrate that DU at pH 7 can
induced oxidative DNA damage in the absence of significant alpha particle
decay, but also suggest that DU can induce carcinogenic lesions, e.g. oxidative
DNA lesions, through interaction with a cellular oxygen species.
[Miller200207JIBv91n1p246] (PMID: 12121782 [PubMed - indexed for MEDLINE]).
10. Depleted Uranium-uranyl
chloride induces apoptosis in mouse J774 macrophages, by J. F. Kalinich,
et al., Applied Cellular Radiobiology Department Armed Forces Radiobiology
Research Institute,
Depleted uranium entering the body as a result of
inhalation or embedded fragments becomes associated to a great extent with
macrophages. As part of our continuing studies on the health effects of internalized
depleted uranium, we investigated the effect of soluble depleted uranium-uranyl
chloride on the mouse macrophage cell line, J774. Using a cytochemical staining
protocol specific for uranium, we found that uranium uptake by the macrophages
increased in a time-dependent manner. Treatment with 1, 10, or 100 microM
depleted uranium-uranyl chloride resulted in decreased viability of the J774
cells within 24 h. Flow cytometric analysis of the treated cells with annexin V
showed the translocation of phosphatidylserine from the inner face of the
plasma membrane to the outer surface indicating the loss of phospholipid
symmetry and the beginning of the apoptotic process. Significant differences in
annexin V labeling between control cells and cells treated with 100 microM
depleted uranium-uranyl chloride were apparent within 2 h. Other events
associated with apoptosis, including morphological changes and DNA
fragmentation, were also apparent after depleted uranium-uranyl chloride
treatment. These results suggest that the uptake and concentration of soluble
depleted uranium by macrophages initiates events that results in the apoptotic
death of these cells.
[Kalinich200209Tv179n1p105]. (PMID: 12204547 [PubMed - indexed for MEDLINE]).
11. Genomic
instability in human osteoblast cells after exposure to depleted uranium:
delayed lethality and micronuclei formation, by A. Miller, et
al., Applied Cellular Radiobiology
Department Armed Forces Radiobiology Research Institute,
It is known that radiation can induce a transmissible persistent destabilization of the genome. We have established an in vitro cellular model using HOS cells to investigate whether genomic instability plays a role in depeleted uranium (DU)-induced effects. Transmissible genomic instability, manifested in the progeny of cells exposed to ionizing radiation, has been characterized by de novo chromosomal aberrations, gene mutations, and an enhanced death rate. Cell lethality and micronuclei formation were measured at various times after exposure to DU, Ni, or gamma radiation. Following a prompt, concentration dependent acute response for both endpoints, there was de novo genomic instability in progeny cells. Delayed reproductive death was observed for many generations (36 days, 30 population doublings) following exposure to DU, Ni, or gamma radiation. While DU stimulated delayed production of micronuclei up to 36 days after exposure, levels in cells exposed to gamma-radiation or Ni returned to normal after 12 days. There was also a persistent increase in micronuclei in all clones isolated from cells that had been exposed to nontoxic concentrations of DU. While clones isolated from gamma-irradiated cells (at doses equitoxic to metal exposure) generally demonstrated an increase in micronuclei, most clonal progeny of Ni-exposed cells did not. These studies demonstrate that DU exposure in vitro results in genomic instability manifested as delayed reproductive death and micronuclei formation.
[Miller200300JERv64n2p247]. (PMID: 12500809 [PubMed - indexed for MEDLINE]).
12. Chromosome aberration analysis in peripheral lymphocytes
of Gulf War and Balkans War veterans, by H. Schroder, et al., Center of Environmental Research and
Technology,
Chromosome
aberrations and sister chromatid exchanges (SCEs) were determined in standard peripheral
lymphocyte metaphase preparations of 13 British Gulf War veterans, two veterans
of the recent war in the Balkans and one veteran of both wars. All 16
volunteers suspect exposures to depleted uranium (DU) while deployed at the two
different theatres of war in 1990 and later on. The
[Schroder200303RPDv103n3p211]. ( PMID: 12678382 [PubMed - in process]).
13. Uranyl acetate causes DNA single strand breaks in vitro
in the presence of ascorbate (Vitamin C), by M.
Yazzie, et al., Department of Chemistry,
Uranium is a radioactive heavy metal with isotopes that decay on the geological time scale. People are exposed to uranium through uranium mining, processing, the resulting mine tailings, and the use of depleted uranium in the military. Acute exposures to uranium are chemically toxic to the kidney; however, little is known about chronic exposures, for example, if there is a direct chemical genotoxicity of uranium. The hypothesis that is being tested in the current work is that hexavalent uranium, as uranyl ion, may have a chemical genotoxicity similar to that of hexavelent chromium. In the current study, reactions of uranyl acetate (UA) and ascorbate (vitamin C) were observed to produce plasmid relaxation in pBluescript DNA. DNA strand breaks increased with increasing concentrations of a 1:1 reaction of UA and ascorbate but were not affected by increasing the ration of ascorbate. Plasmid relaxation was inhibited by coincubation of reactions with catalase but not by coincubation with the radical scavengers mannitol, sodium azide, or 5,5-dimethyl-1-pyrroline-N-oxide. Reactions of UA and ascorbate monitored by (1)H NMR spectroscopy showed formation of a uranyl ascorbate complex, with no evidence of a dehyroascorbate product. A previous study inferred that hydroxyl radical formation was responsible for oxidative DNA damage in the presence of reactions of uranyl ion, hydrogen peroxide, and ascorbate [Miller et al. (2002) J. Bioinorg. Chem. 91, 246-252]. Current results, in the absence of added hydrogen peroxide, were not completely consistent with the interpretation that strand breaks were produced by a Fenton type generation of reactive oxygen species. Data were also consistent with the interpretation that a uranyl ascorbate complex was catalyzing hydrolysis of the DNA-phosphate backbone, in a manner similar to that known for the lanthanides. These data suggest that uranium may be directly genotoxic and may, like chromium, react with DNA by more than one pathway.
[Yazzie200304CRTv16n4p524]. (PMID: 12703969 [PubMed - in process]).
14. Effect of the militarily-relevant
heavy metals, depleted uranium and heavy metal tungsten-alloy on gene
expression in human liver carcinoma cells (HepG2), by A. Miller, et al., Applied
Cellular Radiobiology Department Armed Forces Radiobiology Research Institute,
Depleted uranium (DU) and heavy-metal
tungsten alloys (HMTAs) are dense heavy-metals used primarily in military
applications. Chemically similar to natural uranium, but depleted of the higher
activity 235U and 234U isotopes, DU is a low specific activity, high-density
heavy metal. In contrast, the non-radioactive HMTAs are composed of a mixture
of tungsten (91-93%), nickel (3-5%), and cobalt (2-4%) particles. The use of DU
and HMTAs in military munitions could result in their internalization in
humans. Limited data exist however, regarding the long-term health effects of
internalized DU and HMTAs in humans. Both DU and HMTAs possess a tumorigenic
transforming potential and are genotoxic and mutagenic in vitro. Using
insoluble DU-UO2 and a reconstituted mixture of tungsten, nickel, cobalt
(rWNiCo), we tested their ability to induce stress genes in thirteen different
recombinant cell lines generated from human liver carcinoma cells (HepG2). The
commercially available CAT-Tox (L) cellular assay consists of a panel of cell
lines stably transfected with reporter genes consisting of a coding sequence
for chloramphenicol acetyl transferase (CAT) under transcriptional control by
mammalian stress gene regulatory sequences. DU, (5-50 microg/ml) produced a
complex profile of activity demonstrating significant dose-dependent induction
of the hMTIIA FOS, p53RE, Gadd153, Gadd45, NFkappaBRE, CRE, HSP70, RARE, and
GRP78 promoters. The rWNiCo mixture (5-50 microg/ml) showed dose-related
induction of the GSTYA, hMTIIA, p53RE, FOS, NFkappaBRE, HSP70, and CRE
promoters. An examination of the pure metals, tungsten (W), nickel (Ni), and
cobalt (Co), comprising the rWNiCo mixture, demonstrated that each metal
exhibited a similar pattern of gene induction, but at a significantly decreased
magnitude than that of the rWNiCo mixture. These data showed a synergistic
activation of gene expression by the metals in the rWNiCo mixture. Our data
show for the first time that DU and rWNiCo can activate gene expression through
several signal transduction pathways that may be involved in the toxicity and
tumorigenicity of both DU and HMTAs.
[Miller200401MCBv255n1to2p247]. (PMID:
14971665 [PubMed - in process]).
15. Transcriptomic
and proteomic responses of human renal HEK293 cells to uranium toxicity,
by Prat O, et al., Service de
Biochimie post-genomique et Toxicologie Nucleaire, F-30207 Bagnols-sur-Ceze,
France. odette.prat@cea.fr . Proteomics
Vol. 5 (1), Jan. 2005 (pp. 297-306).
The
industrial use of uranium, in particular depleted uranium, has pin-pointed the
need to review its chemical impact on human health. Global methodologies,
applied to the field of toxicology, have demonstrated their applicability to
investigation of fine molecular mechanisms. This report illustrate the power of
toxicogenomics to evaluate the involvement of certain genes or proteins in
response to uranium. We particularly show that 25% of modulated genes concern
signal transduction and trafficking, that the calcium pathway is heavily
disturbed and that nephroblastomas-related genes are involved (WIT-1, STMN1,
and STMN2). A set of 18 genes was deregulated whatever the concentration of
toxicant, which could constitute a signature of uranium exposure. Moreover, a
group of downregulated genes, with corresponding disappearing proteins (HSP90,
14-3-3 protein, HMGB1) in two-dimensional polyacrylamide gel electrophoresis
(2-D PAGE), are good candidates for use as biomarkers of uranium effects. These
results reveal a cross-checking between transcriptomic and proteomic
technologies. Moreover, our temporal gene expression profiles suggest the
existence of a concentration threshold between adaptive response and severe
cell deregulation. Our results confirm the involvement of genes already
described and also provide new highlights on cellular response to uranium. [Prat200501Pv5n1p297](PMID: 15672453 [PubMed - in
process]).
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